Antibody Specificity: What's the problem? The Antibody Society - - PowerPoint PPT Presentation

Antibody Specificity:
 What's the problem?

The Antibody Society Webcast series – Antibody Validation #1 Andreas Plückthun University of Zurich

Antibodies are known to be specific.
 So how can there be a problem?

The main reason:

• They have not been checked

for specificity

• Specificity cannot be assumed,

but must be experimentally verified What causes non-specific binding, and absence of specific binding?

• 1. Protein surfaces always bind

several things

• 2. Antigens can be in various

conformations, which present different surfaces

• 3. Composition of an antibody

solution may not be what you think it is

Fundamental properties of proteins:
 They bind one another!

• Proteins (antigens, antibodies) have the

intrinsic property of interacting with

• ther proteins
• through adventitious hydrophobic patches
• though adventitious residues that can

make hydrogen bonds

• Since antibodies are proteins, they

cross-react with proteins (antigens) unrelated to their antigens – albeit at very different affinities

Types of antibodies

Polyclonal antibodies:

• Popular, because they are cheap
• taken directly from serum
• And can give strong signals
• they take advantage of many epitopes
• they can bind bivalently in many
• rientations

Types of antibodies

Polyclonal antibodies But:

• There are always antibodies in the

antisera that crossreact with other components

• The composition of two antisera will

never be the same

• It is impossible to reproduce results

from polyclonal sera

Types of antibodies

Monoclonal antibodies Popular, because they are believed to be automatically super-specific

Types of antibodies

Monoclonal antibodies But:

• They can also crossreact with other

proteins

• They may detect other proteins better

than the desired target

• A “monoclonal antibody” is not

necessarily monoclonal !

Types of antibodies

Monoclonal antibodies But

• A monoclonal antibody can also

crossreact with other proteins

The expected case: related proteins "Legitimate crossreactivity" antibody antigens desired undesired closely related protein

Types of antibodies

Monoclonal antibodies But

• A monoclonal antibody can also

crossreact with other proteins

• If not checked properly, it may detect
• ther proteins better than the desired
• ne

The (perhaps) unexpected case: unrelated proteins

• may be unfolded, "sticky"
• may have only few epitopes

"Illegitimate crossreactivity" antibody antigens

Types of antibodies

Monoclonal antibodies But

• A monoclonal antibody can also

crossreact with other proteins

• If not checked properly, it may detect
• ther proteins better than the desired
• ne

The antibody may even adapt to

• ther targets!

"Illegitimate crossreactivity" antibody antigens

Types of antibodies

Monoclonal antibodies But

• A monoclonal antibody can also

crossreact with other proteins

• If not checked properly, it may detect
• ther proteins better than the desired
• ne
• A monoclonal antibody may not even

be monoclonal

Frequently (one third!):

• expression of more than
• ne allele in B-cell
• fusion of more than one B-

cell

• additional light chains from

myeloma fusion partner

Types of antibodies

Monoclonal antibodies Yet another problem:

• As long as the sequence of the antibody

has not be determined, you cannot know whether two antibodies are the same

• Manufacturers sell to each other (same

antibody, different label)

• Manufacturers produce a new lot, maybe

different composition

• It may be impossible to reproduce an

experiment

Frequently (one third!):

• expression of more than
• ne allele in B-cell
• fusion of more than one B-

cell

• additional light chains from

myeloma fusion partner

Types of antibodies

Recombinant antibodies The sequence is known. It can be reproduced forever, the antibody is "immortal" Of course, quality control still has to be done as for every antibody! ➔ By most experts, recombinant technologies are seen as the future

Immunization DNA isolation Synthetic DNA library Display technologies Selection for specificity Quality control Sequence determination

Types of "antibodies"

Recombinant affinity reagents The antibody itself has become dispensable Affinity reagents can be used that are much more stable than antibodies ➔ Other non-antibody scaffolds These can be produced much more cheaply ➔ By most experts, recombinant technologies are seen as the future

Synthetic DNA library Display technologies Selection for specificity Quality control Sequence determination

Types of antigens

Denatured (unfolded proteins)

• usually expose hydrophobic

residues, become more "sticky"

• usually need to be kept in solution

by detergent (SDS), or denaturant (urea, GdnHCl)

Folded proteins

• usually the cellular state
• usually more soluble

Types of antigens

Denatured (unfolded proteins)

• after SDS-Gel electrophoresis

(Western blots)

• after proteolytic digestion
• after tissue fixation (antigen

"retrieval" with a microwave oven!)

Folded proteins

• in cell extracts (pull-down assays)
• on the cell surface (FACS experiments)

Types of antigens

• Many conditions can denature a protein:
• antibodies that recognize the native state no longer bind
• Heat, shaking (=foam), loss of ligands, loss of metals, loss of

subunits,...

Types of antigens

Antibodies can recognize linear epitopes, which will only be accessible in a denatured protein, or in peptide digest

• residues that are close in sequence

but may be hidden in interior

Antibodies can recognize conformational epitopes, which will

• nly be accessible in the folded

protein

• residues that are typically on the

surface, but far apart in sequence

Types of antigens

Most antibodies can only recognize either the folded or the unfolded state!

Types of antigens

Most antibodies can only recognize either the folded or the unfolded state! Most antibodies can thus only be used only for

• either Western blots, IHC (unfolded state recognition)
• or FACS, pull-downs (native state recognition)

Types of antigens

Most antibodies can only recognize either the folded or the unfolded state!

folded

Types of antigens

Most antibodies can only recognize either the folded or the unfolded state!

unfolded

Types of antigens

Most antibodies can only recognize either the folded or the unfolded state!

rather flat groove-like pocket (or peptide side chain)

Types of antigens

Quality-controlling antibodies is by definition application specific!

• You must check antigen recognition in the state of the antigen that will

be used later

• Cross-reactivity will also depend on context:
• other denatured proteins
• other cell components?
• non-proteins contaminants?

Types of antigens

What about ELISA:

• In order to bind to polystyrene, at least part of the protein must

denature!

• Small proteins will almost certainly denature
• Most peptides will not even bind (can be biotinylated)
• Large proteins: often, only one domain denatures, the rest remains
• folded. (Also true for many IgGs themselves)

Types of antigens

What about Immunohistochemistry:

• Antigens are typically crosslinked, epitopes are blocked
• Antigen "retrieval" (heat) denatures the antigen
• Only a small subset of epitopes is suitable for IHC
• It is still very difficult to mimic the "IHC conformation" in vitro, and

thus to test it outside an IHC experiment.

Types of antigens

Are there antibodies can work both in several applications?

• Yes, but only if they are made against a piece
• f the protein which is always unfolded, e.g.

termini (tails) receptors

Types of antigens

Are there antibodies can work both in several applications?

• Yes, polyclonal antibodies
• BUT: they come with the very high price of

cross-reactivities almost impossible to control.

Summary

• 1. Cross-reactivity of antibodies is to be expected. Therefore, it

must be checked

• 2. Monoclonal antibodies are not specific by definition. They must

be checked

• 3. Cross-reactivity is application-specific
• 4. Recombinant antibodies are defined, identifiable and

distinguishable by their sequence – unlike conventional monoclonal antibodies, whose sequence is not known.
 
 But recombinant antibodies must undergo the same checks for cross-reactivity

Antibody Specificity:
 What's the problem?

The Antibody Society Webcast series – Antibody Validation #1 Andreas Plückthun University of Zürich

Next Webcast in Antibody Validation: a 9-part series

1. Andreas Pluckthun : Antibody Specificity: What's the problem? 2. Glenn Begley : Antibodies and the reproducibility crisis in biological science Cecilia Williams : The Erß story – is your antibody like this? 3. Jan Voskuil : Beware the supplier OEM Andy Chalmers : Finding antibodies in the Antibody Databases 4. Anita Bardowski : Which antibody are you looking for? The RRID Jan Voskuil : Points to note on the supplier datasheets 5. Giovanna Roncador: : Correct positive and negative controls in validation 6. Aldrin Gomes : Standard technology: “even” Western blots are non-trivial Jim Trimmer : IHC issues in brain sciences 7. Travis Hardcastle : Cell KO technology Alejandra Solache : Validating Antibodies with KO technology 8. Mike Taussig : Validating antibodies using array technologies Fridjhof Lund-Johansen : Mass spectroscopy for mass validation 9. Andrew Bradbury : Why publish sequences? Andreas Pluckthun : What are the coming alternatives ?

Validation of 
 Commercial tool Antibodies

Antibody Specificity: What's the problem?

The Antibody Society Webcast series – Antibody Validation #1 Presented by Andreas Plückthun

Produced and Directed by Simon L. Goodman Production Manager Fran Breden Writen by Simon Goodman https://www.antibodysociety.org/

Validation of Commercial Tool Antibodies

An Antibody Society Webcast series https://www.antibodysociety.org/

Administrative Support: Dr. Fran Breden and Dr. Mini Muralidharan Executive Director: Dr. Jan Reichert

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